Covalent attachment of biologically active compounds to water-soluble polymers is one method for alteration and control of biodistribution, pharmacokinetics, and often, toxicity for these compounds (Duncan, R. and Kopecek, J. (1984) Adv. Polym. Sci. 57:53-101). Many water-soluble polymers have been used to achieve these effects, such as poly(sialic acid), dextran, poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), poly(N-vinylpyrrolidone) (PVP), poly(vinyl alcohol) (PVA), poly(ethylene glycol-co-propylene glycol), poly(N-acryloyl morpholine (PAcM), and poly(ethylene glycol) (PEG) (Powell, G. M. (1980) Polyethylene glycol. In R. L. Davidson (Ed.) HANDBOOK OF WATER SOLUBLE GUMS AND RESINS. McGraw-Hill, New York, chapter 18). PEG possess an ideal set of properties: very low toxicity (Pang, S. N. J. (1993) J. Am. Coll. Toxicol. 12: 429-456) excellent solubility in aqueous solution (Powell, supra), low immunogenicity and antigenicity (Dreborg, S. and Akerblom, E. B. (1990) Crit. Rev. Ther. Drug Carrier Syst. 6: 315-365). PEG-conjugated or “PEGylated” protein therapeutics, containing single or multiple chains of polyethylene glycol on the protein, have been described in the scientific literature (Clark, R., et al. (1996) J. Biol. Chem. 271: 21969-21977; Hershfield, M. S. (1997) Biochemistry and immunology of poly(ethylene glycol)-modified adenosine deaminase (PEG-ADA). In J. M. Harris and S. Zalipsky (Eds) Poly(ethylene glycol): Chemistry and Biological Applications. American Chemical Society, Washington, D.C., p 145-154; Olson, K., et al. (1997) Preparation and characterization of poly(ethylene glycol)ylated human growth hormone antagonist. In J. M. Harris and S. Zalipsky (Eds) Poly(ethylene glycol): Chemistry and Biological Applications. American Chemical Society, Washington, D.C., p 170-181).
Conjugated proteins have numerous advantages over their unmodified counterparts. For example, PEG-modification has extended the plasma half-life of many proteins (Francis, G. E., et al. (1992) PEG-modified proteins. In: STABILITY OF PROTEIN PHARMACEUTICALS: in vivo PATHWAYS OF DEGRADATION AND STRATEGIES FOR PROTEIN STABILIZATION (ed. by T. J. Ahern and M. Manning). Plenum Press, New York). The basis for this increase involves several factors. The increased size of the PEG-modified conjugate reduces the glomerular filtration when the 70 kD threshold is exceeded (Futertges, F. and Abuchowski, A. (1990) J. Controlled Release 11: 139-148). There is also reduced clearance by the reticuloendothelial system via both carbohydrate receptors and protein-receptor interactions (Beauchamp, C. O., et al. (1983) Anal. Biochem. 131: 25-33). Reduced proteolysis (Chiu, H. C., et al. (1994) J. Bioact. Comp. Polym. 9:388-410) may also contribute to an enhanced half-life. Antigenicity and immunogenicity are also reduced (Nucci, M. L., et al. (1991) Adv. Drug Del. Rev. 6: 133-151), and this accounts for reduction in life-threatening reactions after repeated dosing. The combination of all these factors leads to increased bioavailability in vivo (Katre, N. V., et al. (1987) PNAS USA 84:1487-1491; Hershfield, M. S., et al. (1987) New England Journal of Medicine 316: 589-596), and this is potentially very important in the use of PEG-cytokine adducts as pharmacological agents. Dose can be reduced (to alleviate toxicity) and more convenient schedule of dosing can be developed.
IL-18 is a non-glycosylated monomer of 18 Kd with a primary structure most closely related to IL-1α of the IL-1β-trefoil subfamily. Murine and human IL-18 cDNA encode a precursor protein consisting of 192 and 193 amino acids, respectively. The homology between human and murine IL-18 is 65%. Pro-IL-18 requires processing by caspases, such as ICE (caspase-1) or caspase-4, into bioactive mature protein (157 amino acids) in order to mediate biologic activity. The activity of IL-18 is mediated via an IL-18 receptor (IL-18R) complex (made up of a binding chain (IL-18Rα) and a signaling chain (IL-18Rβ)). The biological activities of IL-18 that support its therapeutic potential for tumor immunotherapy include induction and production of IFNγ and GM-CSF, enhancement of NK cell cytolytic activity and promotion, and differentiation of naive T cells into Th1 cells. In response to IL-18, cytotoxic T lymphocytes (CTLs) and memory cells are generated that display potent anti-tumor activity. Other regulatory functions include up-regulation of functional Fas ligand (FasL) expression on NK and T cells (suggesting that the IL-18 anti-tumor activity is mediated in part by Fas-FasL interaction, inducing tumor apoptosis); activation of monocytes/macrophage, B cells, and anti-angiogenesis.
IL-18 binding protein (IL-18BP) is a naturally occurring soluble circulating protein that has been recently described as an antagonist of IL-18. IL-18BP bears no significant homology to either IL-18 receptor, in that it contains a single putative Ig domain that bears very limited homology to the third Ig domain of the type II IL-1 receptor. Much greater homology to IL-18BP can be found in a family of proteins encoded by several poxviruses (swinepox, cowpox, variola, molluscum, contagiosum, and ectromelia). Poxviruses encode decoy receptors of many cytokines and these receptors are instrumental in viral avoidance of immune responses. Because IL-18 is one of the early signals leading to IFNγ production by Th1 cells, blocking IL-18 activity by IL-18BP may be involved in down-regulating one of the earliest phases of the immune response. Elevated levels of IL-18BP could be detrimental to the effectiveness of recombinant IL-18 therapy.
As a single agent, a recombinant form of murine IL-18 stimulated the murine immune system, resulting in partial and complete tumor regressions and/or induction of immunological memory in various established tumor models. In combination with chemotherapeutic agents commonly used in the clinical setting, such as topotecan, murine IL-18 demonstrated a synergistic effect, resulting in improved efficacy at the local and/or systemic levels in various established tumor models. Potential biomarkers of IL-18 activity were investigated to correlate with early events of IL-18 mediated immune activation, together with extensive toxicology and pharmacokinetic studies of both murine IL-18 and human IL-18 recombinant forms of IL-18. These pre-clinical data support the clinical development of human IL-18 as a novel form of immunotherapy, or as an adjunct for cancer vaccines, or an adjuvant to cytotoxic agents and other biologicals, such as topotecan and IL-2 respectively, for the treatment of patients suffering from different types of cancers.